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. 2019 Jun 27;9:9309. doi: 10.1038/s41598-019-45797-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Spheroid size, proliferation, viability and cell cycle analysis after nanoparticles exposure (5 and 100 µg/ml of TiO₂ NPs for 72 hours): (A) Spheroid diameter and volume of 10,000 cells spheroids upon NPs exposure was calculated based on phase contrast image analysis by area determination using image J software. Data are shown as mean ± SD of three independent experiments (n = 10). (B) spheroid proliferation during formation and NPs exposure. (C) Annexin V/PI assay captures the different cell death profiles in the spheroids exposed to different concentrations of TiO₂ NPs. (D) cell cycle analysis showing percentages of Go-G1 phase, S and G2/M phase of spheroids exposed to NPs. Bar represents the mean ± SD performed with 4 spheroids in each condition and the analyses were performed in triplicate. * and **P < 0.05 vs control.